Breast Cancer (breast Ca) is a leading cause of death for women in the U.S. and will affect approximately 105 of the present female population. Many studies have demonstrated that 17beta-estradiol (E2) is a critical factor in the genesis of breast Ca and can promote breast Ca cell growth in vivo as well as in vitro in the presence of growth factors. However, the mechanisms underlying regulation of the breast Ca cell cycle by E2 are poorly, if at all, understood. Our recent data suggests that E2 (in the absence of exogenous growth factors) can directly activate cyclin dependent kinase 2 (Cdk2) activity in MCF-7 cells, leading to hyperphosphorylation of retinoblastoma protein (pRb) and induction of S- phase entry. Furthermore, E2 appears to inactivate a Cdk inhibitor (CKI) present in untreated G1 cells. These regulatory effects of E2 in the nanomolar concentration range are inhibited by an excess of steroidal antiestrogen (ICI l82,780). Thus, we hypothesize that E2 directly regulates one or more of the following critical steps in the MCF-7 cell cycle (a) Expression of D cyclin gene(s) and synthesis of D cyclin(s); (b) Activation of Cdk4 kinase activity; (c) Inhibition of a CKI active in G1 phase; and (d) Activation of Cdk2 kinase activity (perhaps via activation of Cdk activating kinase - CAK) leading to pRb hyperphosphorylation and entry into S-phase. We have proposed 3 specific aims to directly test facets of this hypothesis. They are: (1) Studies to investigate whether E2 regulates D- type cyclin synthesis/degradation, in particular that of cyclin D1, and whether Cdk4 kinase activity and/or steady-state protein levels are regulated by E2. (2) We will investigate the possibility that E2 treatment inactivates a Cdk inhibitor present in quiescent MCF-7 cells resulting in Cdk activation and G1 transit. We propose that E2 will be unable to bring about inactivation of putative inhibitors present in ER negative breast Ca cells or other cells lacking ER. (3) Studies under Specific Aim 3 will explore the possibility that E2, directly or indirectly, regulates cyclin D1 promoter activity. Cyclin D1 promoter reporter gene constructs will be expressed in MCF-7 cells and ER negative breast Ca cells. Specific activation of these promoter constructs in response to E2 will be measured, and specific response elements required for this activation will be determined by deletion and mutation experiments. The results of these studies will, for the first time, directly demonstrate how E2 regulates early events in the cell cycle of E2 responsive breast Ca cells.